Coordinated control of renal Ca2+ transport proteins by parathyroid hormone

Kidney International, Vol. 68 (2005), pp. 1708–1721

Coordinated control of renal Ca2+ transport proteins by parathyroid hormone MONIQUE VAN ABEL, JOOST G.J. HOENDEROP, ANNEMIETE W.C.M. VAN DER KEMP, MICHAEL M. FRIEDLAENDER,† JOHANNES P.T.M. VAN LEEUWEN, and RENE´ J.M. BINDELS Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem, Israel; and Department of Internal Medicine, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands

Coordinated control of renal Ca2+ transport proteins by parathyroid hormone. Background. The kidney is one of the affected organs involved in the clinical symptoms of parathyroid hormone (PTH)related disorders, like primary hyperparathyroidism and familial hypocalciuric hypercalcemia. The molecular mechanism(s) underlying alterations in renal Ca2+ handling in these disorders is poorly understood. Methods. Parathyroidectomized and PTH-supplemented rats and mice infused with the calcimimetic compound NPS R-467 were used to study the in vivo effect of PTH on the expression of renal transcellular Ca2+ transport proteins, including the epithelial Ca2+ channel transient receptor potential, vanilloid, member 5 (TRPV5), calbindins, and the Na+ /Ca2+ -exchanger (NCX1). In addition, the effect of PTH on transepithelial Ca2+ transport in rabbit connecting tubule/cortical collecting duct (CNT/CCD) primary cultures was determined. Results. Decreased PTH levels in parathyroidectomized rats or NPS R-467–infused mice, resulted in reduced expression of these proteins, which is consistent with diminished Ca2+ reabsorption, causing the development of the observed hypocalcemia. PTH supplementation of parathyroidectomized rats restored the expression of the renal Ca2+ transport machinery and serum Ca2+ levels, independent of serum 1,25-dihydroxyvitamin D 3 levels and renal vitamin D or Ca2+ -sensing receptor mRNA abundance. Inhibition of the PTH-stimulated transepithelial Ca2+ transport by the TRPV5specific inhibitor ruthenium red reduced the PTH-stimulated expression of calbindin-D 28K and NCX1 in rabbit CNT/CCD primary cultures. Conclusion. PTH stimulates renal Ca2+ reabsorption through the coordinated expression of renal transcellular Ca2+ transport proteins. Moreover, the PTH-induced stimulation is enhanced by the magnitude of the Ca2+ influx through the gatekeeper

† Deceased. 2+

Key words: ECaC, TRPV5, TRPV6, PTH, Ca -sensing receptor, calcimimetic, NPS R-467, ruthenium red. Received for publication January 4, 2005 and in revised form April 20, 2005 Accepted for publication May 23, 2005
C

2005 by the International Society of Nephrology

TRPV5, which in turn facilitates the expression of the downstream Ca2+ transport proteins. Therefore, the renal transcellular Ca2+ transport proteins, including TRPV5, could contribute to the pathogenesis of PTH-related disorders.

Primary hyperparathyroidism is a common endocrine disorder characterized by elevated parathyroid hormone (PTH) levels that are inappropriate to the level of serum Ca2+ [1]. This underlines the importance of PTH as an essential component of Ca2+ homeostasis. Secretion of PTH from the parathyroid glands is regulated by the ambient Ca2+ concentration, sensed by the parathyroid Ca2+ -sensing receptor [2]. The Ca2+ -sensing receptor is activated by high serum Ca2+ concentrations and couples to the inhibition of PTH secretion. Inactivating mutations of the Ca2+ -sensing receptor result in familial hypocalciuric hypercalcemia or neonatal severe hyperparathyroidism [3], whereas autosomal-dominant hypocalcemia is caused by activating mutations [4]. These disorders demonstrate the predominant role of the Ca2+ -sensing receptor in controlling parathyroid gland function and, hence, the role of PTH in regulating systemic Ca2+ balance. From the clinical symptoms of these PTH-related disorders, like hypo- or hypercalciuria and renal stone formation, it is clear that also renal Ca2+ handling is affected. Regulation of Ca2+ reabsorption in the kidney is crucial for the maintenance of normal serum Ca2+ levels and occurs via paracellular as well as transcellular routes. Transcellular or active Ca2+ reabsorption is the primary target for regulation by calciotropic hormones, including 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] and PTH. Active Ca2+ reabsorption takes place in the distal convoluted tubule (DCT) and connecting tubule (CNT) of the kidney. Luminal Ca2+ enters these tubular cells via the epithelial Ca2+ channels, transient receptor potential, vanilloid, members 5 and 6 (TRPV5 and TRPV6), is then transported across the cell in

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association with Ca2+ -carrier proteins (i.e., calbindins) and is finally extruded into the blood stream via the Na+ /Ca2+ -exchanger (NCX1) and the plasma membrane Ca2+ -ATPase (PMCA1b) [5–8]. PTH receptors have been detected throughout the kidney, as well as in the actively Ca2+ transporting tubules DCT and CNT [9, 10]. Therefore, the contribution of these Ca2+ transport proteins to the altered Ca2+ handling in PTH-related disorders seems likely. Treatment of primary hyperparathyroidism has been limited to surgical ablation (parathyroidectomy) of the affected glands. A new approach for treating primary hyperthyroidism is to target the mechanisms that regulate the secretion of PTH. The Ca2+ -sensing receptor is the first step in this process. In the last decade synthetic compounds, referred to as calcimimetic compounds, have been developed [11, 12]. Type I calcimimetics are full agonists of the Ca2+ -sensing receptor, whereas type II calcimimetics are small organic compounds that, upon binding to the Ca2+ -sensing receptor, enhance the sensitivity of the Ca2+ -sensing receptor to Ca2+ in an allosteric fashion, thereby altering PTH secretion by the parathyroid glands. In this way, the type II calcimimetic compounds may provide a novel therapy for treating hyperparathyroidism [11, 12]. The present study aims to gain insight into the molecular mechanism(s) underlying the alterations in renal Ca2+ handling in PTH-related disorders. To this end, the in vivo effect of PTH on the expression of proteins involved in renal transcellular Ca2+ reabsorption was examined. Here, we demonstrated that PTH affects renal Ca2+ handling through the coordinated regulation of the expression of renal transcellular Ca2+ transport proteins. Moreover, the PTH-induced increase in Ca2+ influx through TRPV5 facilitates the expression of downstream Ca2+ transport proteins, thereby further emphasizing the gatekeeper function of TRPV5.

ad libitum. After acclimatization, mice were divided in four groups of five animals each. Mice were anesthetized and an Alzet model 1007D osmotic minipump (Durect Corporation) was implanted subcutaneously. The pump infused the calcimimetic compound NPS R-467 (NPS Pharmaceuticals, Salt Lake City, UT, USA) at a dose of 10, 30, or 100 lmol per kg body weight per day or vehicle solution [45% (wt/vol) aqueous solution of 2-hydroxypropyl-b-cyclodextrin]. At the end of the treatment periods, animals were sacrificed, and blood and kidney samples were taken. The animal ethics board of the University Medical Center Nijmegen and Hadassah University Hospital Jerusalem approved all animal experimental procedures.

METHODS Animals In experiment 1, male Sabra rats, weighing 250 g, had free access to normal rat chow and water. After acclimatization, rats were either sham-operated (N = 4) or parathyroidectomized (N = 8) by cauterization. After 7 days, Alzet osmotic minipumps were implanted subcutaneously (model 2001) (Durect Corporation, Cupertino, CA, USA). Sham-operated rats received vehicle solution [2% (wt/vol) cysteine HCl] and parathyroidectomized rats received either vehicle solution or bovine PTH 1-34 (Sigma Chemical Co., St. Louis, MO, USA) at a rate of 0.25 U PTH 1-34/hour (parathyroidectomized + PTH) for 7 days. In experiment 2, Male C57BL6 mice, 8 weeks of age, were fed standard chow and given water

Determination of transepithelial Ca2+ transport

Primary cultures of rabbit kidney CNT/cortical collecting duct (CCD) Rabbit kidney CNT/CCD cells were immunodissected from New Zealand white rabbits (5 weeks of age) with monoclonal antibody R2G9 and set in primary culture on permeable filter supports (0.33 cm2 ) (Corning-Costar, Cambridge, MA, USA) as described previously in detail [13]. The culture medium was a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium (Gibco, Paisley, UK) supplemented with 5% (vol/vol) decomplemented fetal calf serum (FCS), 10 lg/mL ciproxin, 10 lg/mL nonessential amino acids (Gibco), 5 lg/mL insulin, 5 lg/mL transferrin, 50 nmol/L hydrocortisone, 70 ng/mL prostaglandin E 1 (PGE 1 ), 50 nmol/L Na 2 SeO 3 , 5 pmol/L triiodothyronine, and 5 lmol/L indomethacin, equilibrated with 5% CO 2 95% air at 37◦ C. Two days after seeding, the cells were incubated for 120 hours with and without 100 nmol/L bovine PTH 1-34 (Sigma Chemical Co.) at the apical (total volume 100 lL) and basolateral (total volume 600 lL) compartment, whereas 10 lmol/L ruthenium red was added only to the apical side. Transport assays were performed with confluent monolayers 7 days after seeding the cells.

Confluent monolayers of primary cultures were washed and preincubated in a physiologic salt solution containing (mmol/L) 140 NaCl, 2 KCl, 1 K 2 HPO 4 , 1 KH 2 PO 4 , 1 MgCl 2 , 1 CaCl 2 , 5 glucose, 5 L-alanine, 10 Hepes/Tris (pH 7.4), and 5 lmol/L indomethacin for 15 minuts at 37◦ C, adding 10 lmol/L of the TRPV5mediated Ca2+ -influx blocker ruthenium red [14] to the apical side. Subsequently, the monolayers were incubated with 100 nmol/L PTH and 10 lmol/L ruthenium red for a further 90 minutes to measure transepithelial Ca2+ transport. At the end of the incubation period, 25 lL samples were collected in duplicate from the apical compartment and assayed for Ca2+ concentration using a colorimetric assay kit (Boehringer, Mannheim, Germany). Under the

outlined experimental conditions, these polarized renal cells exhibit transcellular Ca2+ transport, of which the net apical-to-basolateral Ca2+ flux is linear with time for at least 3 hours [13, 15]. Ca2+ reabsorption is expressed in nmol/hour−1 /cm−2 .

TRPV5 CaBP NCX1

VDR

CaSR

R M R M Rb Rb Rb

Reverse primer

5 -GCGAATATCATGGCTTGTAACCA-3 5 -GCAAAGATCATGGCTTGTAACCA-3 5 -TGTCCACACAGCGTTTGA-3 5 -TGTCCACGCAGCGTTTGA-3 5 -CCTTGTCTGAGCACTTGAATGC-3 5 -CCACACATTTTGATTCCCTGAA-3 5 -TTTCCGTGTTGACTTCATGCA-3

Forward primer

5 -CTTTCCTATCCATTTTGGAGTAGCA-3 5 -CTTTCCTATCCATTTTGGAGTAGCA-3 5 -AATGGAGATTGCCGCATCAC-3 5 -AATGGAGATTGCCGCATCAC-3 5 -CGGGTTGAGAACCATCATGAC-3 5 -GATGGGAAGCTGGAATTAACTGA-3 5 -GGGCTGGCAACATTTTAAAGAG-3

Immunohistochemistry Kidney tissue was cut into pieces, placed in 1% (wt/vol) periodate-lysine-paraformaldehyde fixative for 2 hours at room temperature, and incubated overnight at 4◦ C in phosphate-buffered saline (PBS) containing 15% (wt/vol) sucrose. Subsequently, kidney tissues were frozen in liquid nitrogen and 7 lm sections were cut for the staining procedure. For detection of TRPV5 abundance or colocalization of TRPV5 and Ca2+ -sensing receptor, kidney sections were stained with guinea pig antiTRPV5 antiserum (1:50) as described previously [19] and rabbit antihuman Ca2+ -sensing receptor antibody (1:500) (antiserum 4641, a gift from NPS Pharmaceuticals).

Gene

RNA isolation and quantitative polymerase chain reaction (PCR) Total RNA from kidney and primary CNT/CCD cells was isolated using Trizol Reagent (Gibco BRL, Life Technologies, Breda, The Netherlands) according to the manufacturer’s protocol. Total DNAse-treated RNA (2 lg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (MMLV-RT) (Gibco BRL) as described previously [18]. Expression of TRPV5, TRPV6, calbindin-D 28K , calbindin-D 9K , NCX1, PMCA1b, Ca2+ -sensing receptor, and vitamin D receptor mRNA, as well as mRNA levels of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT), as an endogenous control, were determined by quantitative real-time PCR on an ABI Prism 7700 Sequence Detection System (PE Biosystems, Rotkreuz, Switserland). The primer and probe sequences of Ca2+ sensing receptor, vitamin D receptor, TRPV5, calbindinD 28K , and NCX1 are depicted in Table 1. Other sequences have been described previously [19, 20].

Table 1. Sequences of primers and Taqman probes for real-time quantitative polymerase chain reaction (PCR)

Serum Ca2+ concentrations were analyzed using a colorimetric assay kit as described previously [16]. Serum phosphorus levels were measured on a Hitachi autoanalyzer (Hitachi Corp., Tokyo, Japan). PTH levels were determined using either a rat PTH (1-34) immunoradiometric assay or a mouse intact PTH enzyme-linked immunosorbent assay (ELISA) kit (Immunotopics, San Clemente, CA, USA). 1,25(OH) 2 D 3 was measured by immunoextraction followed by quantitation by 125 I-radioimmunoassay (RIA) (IDS, Boldon, UK) [17].

Probe

Analytical procedures

Abbreviations are: CaSR, Ca2+ -sensing receptor; VDR, vitamin D receptor; TRPV5, transient receptor potential, vanilloid, member 5; CaBP, calbindin-D 28K ; NCX1, Na+ /Ca2+ -exchanger; R, rat; M, mouse; Rb, rabbit. Sequences of the other target genes used are as described previously [19, 20]. PCR primers and fluorescent probes (5 -FAM to 3-TAMRA) were designed using the computer program Primer Express (Applied Biosystems, Branchburg, NJ, USA) and purchased from Biolegio (Malden, The Netherlands).

van Abel et al: PTH-dependent regulation of renal TRPV5

5 -CGGAAGTTATACCTAATGCACTCCACAGACTCT-3 5 -CGGAAGTTATACCTGATGCACTCCACAGACTCT-3 5 -AGGACAACCGGCGACACTGCCA-3 5 -AGGACAACCGGCGACACTGCCA-3 5 -TCCTCTGCGAGTGCTTCGCTATGTGG-3 5 -ATGGCCAGGTTACTACCAGTGCAAGAGAATTT-3 5 -AGCTGACCAAGCTAGGAAGGCTGTCAGC-3

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Table 2. Effect of parathyroidectomy and parathyroid hormone (PTH) supplementation on serum parameters in Sabra ratsa

PTH pg/mL Ca2+ mmol/L Phosphorus mmol/L 1,25(OH) 2 D 3 pmol/L

Sham

Parathyroidectomy

Parathyroidectomy + PTH

43.5 ± 14.7 2.89 ± 0.06 1.59 ± 0.06 375 ± 9

6.1 ± 2.9b 1.61 ± 0.07c 2.15 ± 0.04c 261 ± 29b

14.4 ± 4.6 2.51 ± 0.04d 1.82 ± 0.10d 234 ± 13b

Sham, sham-operated; parathyroidectomy + PTH, parathyroidectomy supplemented with 0.25 U PTH (1-34)/hour. Data are presented as mean ± SE (N = 4). P < 0.05 versus sham. c < P 0.05 versus sham and parathyroidectomy + PTH. d < P 0.05 versus sham and parathyroidectomy. a

b

To visualize TRPV5 and Ca2+ -sensing receptor, sections were stained with goat antiguinea Alexa 488–conjugated anti-IgG (1:300) (Sigma Chemical Co.) and goat antirabbit Alexa 594–conjugated anti-IgG (1:300) (Sigma Chemical Co.), respectively. Sections were visualized by confocal laser scanning microscopy (MRC-1024) (BioRad, Richmond, CA, USA). To quantify TRPV5 protein expression, digital images of each kidney section were taken with a Zeiss Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY, USA) and the integrated optical density was measured by computer analysis with the ImagePro Plus version 3.0 software (Media Cybernetics, Silver Spring, MD, USA). Immunoblotting For protein analysis, frozen kidney tissue and the cultured cells were homogenized in ice-cold solubilization buffer as previously described [21]. Total kidney protein fractions (10 lg) or cell protein fractions (4 lL) were separated on 12% or 16.5% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and blotted to polyvinylidine difluoride (PVDF)-nitrocellulose membranes (Immobilon-P) (Millipore Corporation, Bedford, MA, USA). Blots were incubated with calbindin-D 28K antibody (1:10,000) (Sigma Chemical Co.), calbindin-D 9K antibody (1:3000) (Swant, Bellinzona, Switzerland), or Na+ /K+ -ATPase antibody (1:10,000) (kindly provided by J.B. Koenderink, Department of Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) [22] and thereafter with peroxidase-conjugated goat antirabbit antibody (1:2000) (Sigma Chemical Co.). Immunoreactive protein was detected using the enhanced chemiluminescence (ECL) method as described by the manufacturer (Amersham, Buckinghamshire, UK). Protein expression was quantified by computer-assisted densitometry with the use of the Image-Pro Plus version 3.0 software (Media Cybernetics). Statistical analysis Values are expressed as mean ± SE. Statistical significance of differences between groups was determined by analysis of variance (ANOVA) followed by pair-wise comparisons using the method of least significant differ-

ence. An unpaired t test was used for comparisons between control and 100 lmol/kg body weight/day groups. Differences in means with P < 0.05 were considered statistically significant. RESULTS Rescue of parathyroidectomy-induced hypocalcemia by PTH supplementation in rats Surgical removal of the parathyroid glands resulted in the reduction of serum PTH levels, whereas serum PTH levels tended to increase after infusion with 0.25 U PTH (1-34)/hour (Table 2). Moreover, parathyroidectomy caused a severe hypocalcemia and hyperphosphatemia, which were partially restored by PTH supplementation (Table 2). In addition, serum 1,25(OH) 2 D 3 levels were decreased in parathyroidectomized rats, but no changes were observed after PTH supplementation (Table 2). Effect of PTH on renal expression of Ca2+ transporters To investigate the effect of PTH on renal Ca2+ handling, the expression of genes encoding Ca2+ transport proteins involved in transcellular Ca2+ reabsorption was examined using quantitative real-time PCR. Surgical removal of the parathyroid glands in rats induced a 70% decrease in TRPV5 mRNA levels, which were completely normalized after PTH infusion (Fig. 1A). In contrast, no significant effects were detected on the expression of TRPV6 mRNA between sham-operated, parathyroidectomy, and parathyroidectomy + PTH rats (Fig. 1B). Similar to TRPV5 expression, mRNA transcripts encoding for calbindin-D 28K were down-regulated in parathyroictomized rats and partially restored in the PTH-supplemented animals (Fig. 1C). The expression of calbindin-D 9K was not measured, because it is not detectable in rat kidney. In addition, parathyroidectomy reduced the expression of NCX1 (Fig. 1D), whereas PMCA1b mRNA levels were not altered (Fig. 1E). No differences were observed in Ca2+ -sensing receptor or vitamin D receptor mRNA levels after parathyroidectomy or subsequent infusion of PTH (Fig. 1F and G). Subsequently, examination of the expression of renal Ca2+ transport proteins by immunohistochemistry revealed a marked decrease in TRPV5 protein

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Fig. 1. Effect of parathyroidectomy and supplementation with parathyroid hormone (PTH) (1-34) on mRNA expression levels of genes encoding Ca2+ transport proteins in rat kidney. Using real-time quantitative polymerase chain reaction (PCR), renal mRNA expression of transient receptor potential, vanilloid, member 5 (TRPV5) (A), TRPV member 6 (TRPV6) (B), calbindin-D 28K (C), Na+ /Ca2+ -exchanger (NCX1) (D), plasma membrane Ca2+ -ATPase (PMCA1b) (E), Ca2+ -sensing receptor (CaSR) (F), and vitamin D receptor (VDR) (G) of the different experimental groups were measured and presented as a ratio to hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression. Sham-operated rats (
); parathyroidectomized rats (
); parathyroidectomizied rats supplemented with 0.25 U PTH (1-34)/hour (). Data are presented as means ± SE (N = 4). ∗ P < 0.05 versus all; # P < 0.05 versus sham.

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Fig. 2. Effect of parathyoidectomy (PTX) and supplementation with parathyroid hormone (PTH) (1-34) on protein expression levels of transient receptor potential, vanilloid, member 5 (TRPV5) in rat kidney. Immunofluorescence staining of kidney cortex sections of sham, parathyroidectomy, and parathyroidectomy + PTH rats showing the abundance of TRPV5 (A), which was quantified by computer analysis of the integrated optical density and expressed in relative percentages (B). Glomeruli (G) are depicted in the images. Immunoblots of samples (10 lg protein each) from homogenates of kidney tissue were labeled with antibodies against calbindin-D 28K (D). Expression of calbindin-D 28K protein was quantified by computer-assisted densitometry analysis and expressed in relative percentages (C). Sham (
), sham-operated rats; parathryoidectomy (
), parathyroidectomized rats + PTH (), parathyroidectomized rats supplemented with 0.25 U PTH (1-34)/hour. Data are presented as means ± SE (N = 4). ∗ P < 0.05 versus sham; # P < 0.05 versus all.

abundance in response to parathyroidectomy, as indicated by the reduced immunopositive staining (Fig. 2A). As observed for TRPV5 mRNA expression, PTH supplementation increased the protein abundance in the parathyroidectomized animals. The corresponding integrated optical density analysis confirmed the significant decline in TRPV5 protein abundance after parathyroidectomy (Fig. 2B). Furthermore, Western blot analysis of calbindin-D 28K consistently demonstrated a down-regulation in the protein abundance in parathyroidectomized rats and a subsequent normalization after PTH infusion (Fig. 2D). Densitometric analysis of the intensity of the immunocomplexes confirmed this decrease in calbindin-D 28K protein expression after parathyroidectomy (Fig. 2C).

NPS R-467 treatment reduced serum PTH and Ca2+ levels in mice Infusion of the calcimimetic compound NPS R-467 for 7 days reduced serum PTH levels. Furthermore, this reduction was associated with a fall in serum Ca2+ concentrations and an increase in serum phosphorus levels. Measurements of circulating 1,25(OH) 2 D 3 levels revealed no effect of NPS R-467 treatment compared with vehicle (Table 3). Reduction in renal Ca2+ transport proteins after NPS R-467 treatment To address the molecular mechanism responsible for the decreased serum Ca2+ levels upon NPS R-467

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van Abel et al: PTH-dependent regulation of renal TRPV5 Table 3. Effect of treatment with NPS R-467 on serum parameters in male C57BL6 micea

Parthyroid hormone pg/mL Ca2+ mmol/L Phosphorus mmol/L 1,25(OH) 2 D 3 pmol/L

Control

10 lmol/kg

30 lmol/kg

100 lmol/kg

20.8 ± 2.5 2.42 ± 0.02 2.07 ± 0.19 309 ± 59

14.6 ± 1.2b 2.34 ± 0.02 2.72 ± 0.09b 326 ± 14

13.0 ± 0.6b 2.26 ± 0.02b 2.72 ± 0.12b 307 ± 10

10.8 ± 1.2b 2.18 ± 0.06b,c 2.71 ± 0.06b 227 ± 27

a Control, treated with vehicle; 10 lmol/kg, treated with NPS R-467 at 10 lmol/kg body weight/day; 30 lmol/kg, treated with NPS R-467 at 30 lmol/kg body weight/day; 100 lmol/kg, treated with NPS R-467 at 100 lmol/kg body weight/day. Data are presented as means ± SE (N = 5). b < P 0.05 versus control. c < P 0.05 versus 10 lmol/kg.

infusion, the expression of genes encoding Ca2+ transport proteins involved in renal transcellular Ca2+ reabsorption was examined. TRPV5 mRNA expression was consistently lower in kidneys of NPS R-467–treated mice compared to controls (Fig. 3A), whereas no differences in the expression of TRPV6 were detected (Fig. 3B). Similar to TRPV5 expression, mRNA levels of both calbindin-D 28K and calbindin-D 9K were down-regulated in response to calcimimetic treatment (Fig. 3C and D). In addition, NPS R-467 treatment inhibited the expression of NCX1 (Fig. 3E), whereas PMCA1b mRNA levels were only down-regulated at 100 lmol/kg body weight/day compared to control animals (Fig. 3F). Infusion with NPS R-467 induced a reduction in the expression of Ca2+ sensing receptor mRNA compared to control mice (Fig. 3G), while no differences were detected in the mRNA expression of vitamin D receptor (Fig. 3H). Subsequently, the reduced mRNA levels of TRPV5 after NPS R-467 treatment were confirmed by immunohistochemical analysis. As shown in Figure 4A, the staining intensity of the TRPV5 protein was remarkably reduced in kidney cortex sections of NPS R-467–treated mice. Figure 4B depicts the corresponding integrated optical density analysis and demonstrated that TRPV5 protein expression is significantly down-regulated. In addition, the immunoblots shown in Figure 4C and E demonstrated a down-regulation of both calbindin-D 28K and calbindinD 9K proteins in the NPS R-467–treated mice compared to controls, which was confirmed by densitometry (Fig. 4D and F). Localization of the Ca2+ -sensing receptor in mouse kidney cortex In addition to the presence in the parathyroid glands, the Ca2+ -sensing receptor is also expressed in kidney. In Figure 5, the distribution of Ca2+ -sensing receptor immunopositive staining in kidney cortex was compared to the TRPV5 localization. The distribution of Ca2+ sensing receptor was most abundant in the basolateral region of the thick ascending limb (Fig. 5A, arrowheads). Importantly, immunopositive staining for Ca2+ -sensing receptor (Fig. 5A, asterisks, and C) was also observed in TRPV5 expressing DCT and CNT (Fig. 5B, aster-

isks, and D). TRPV5 was predominantly localized along the apical domain of these segments, whereas Ca2+ sensing receptor–specific staining revealed a more diffuse pattern. Inhibition of PTH-stimulated transepithelial Ca2+ transport decreased calbindin-D 28K and NCX1 expression To determine the molecular mechanism responsible for the regulation of the Ca2+ transport proteins, primary cultures of the rabbit CNT/CCD grown to confluence on permeable supports were employed. Figure 6A shows that in the absence of any stimulus, these monolayers exhibit a net apical-to-basolateral Ca2+ flux of 43 ± 2 nmol/hour−1 /cm−2 . Treatment with PTH (100 nmol/L) had a stimulatory effect on transepithelial Ca2+ reabsorption (Fig. 6A). Apical addition of the Ca2+ channel blocker ruthenium red (10 lmol/L) completely inhibited basal transepithelial Ca2+ reabsorption and PTH-stimulated Ca2+ transport (Fig. 6A). Furthermore, the stimulatory effect of PTH on Ca2+ transport was accompanied by increased mRNA expression of TRPV5, calbindin-D 28K and NCX1 (Fig. 6B to D). Importantly, this PTH-induced increase in calbindin-D 28K and NCX1 mRNA expression was significantly reduced in monolayers cultured in the presence of ruthenium red, while the mRNA expression of TRPV5 was unaltered after ruthenium red treatment. Immunoblot and densitometric analysis of the intensity of the immunocomplexes of calbindin-D 28K confirmed this decrease in calbindin-D 28K protein expression after ruthenium red treatment using the Na+ /K+ -ATPase as an internal control for equal loading (Fig. 6E and F).

DISCUSSION The present study demonstrated that PTH stimulates renal Ca2+ reabsorption through the coordinated expression of transcellular Ca2+ transport proteins. Moreover, our results indicated that by up-regulating the expression of the TRPV5, the Ca2+ influx is increased, which in turn facilitates the regulation of the down-stream Ca2+ transport proteins. These findings further emphasize the gatekeeper function of TRPV5.

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Fig. 3. Effect of NPS R-467 on mRNA expression levels of genes encoding Ca2+ transport proteins in the kidney. Using real-time quantitative polymerase chain reaction (PCR), renal mRNA expression of transient receptor potential, vanilloid, member 5 (TRPV5) (A), TRPV member 6 (TRPV6) (B), calbindin-D 28K (C), calbindin-D 9K (D), Na+ /Ca2+ -exchanger (NCX1) (E), plasma membrane Ca2+ -ATPase (PMCA1b) (F), Ca2+ -sensing receptor (CaSR) (G), and vitamin D receptor (VDR) (H) of the different experimental groups were measured and presented as a ratio to hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression. Control mice treated with vehicle (
); mice treated with NPS R-467 at 10 lmol/kg body weight/day (
); mice treated with NPS R-467 at 30 lmol/kg body weight/day (
); mice treated with NPS R-467 at 100 lmol/kg body weight/day (). Data are presented as means ± SE (N = 5). ∗ P < 0.05 versus control; # P < 0.05 versus all.

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van Abel et al: PTH-dependent regulation of renal TRPV5

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van Abel et al: PTH-dependent regulation of renal TRPV5

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Fig. 4. Effect of NPS R-467 on protein expression levels of transient receptor potential, vanilloid, member 5 (TRPV5) in the kidney. Immunofluorescence staining of kidney cortex sections of control and treated mice showing the abundance of TRPV5 (A), which was quantified by computer analysis of the integrated optical density and expressed in relative percentages (B). Glomeruli (G) are depicted in the images. Immunoblots of samples (10 lg protein each) from homogenates of kidney tissue were labeled with antibodies against calbindin-D 28K (C) and calbindin-D 9K (E). Expression of calbindin-D 28K and calbindin-D 9K protein was quantified by computer-assisted densitometry analysis and expressed in relative percentages (D and F). Control (
), mice treated with vehicle; treated (), mice treated with NPS R-467 at 100 lmol/kg body weight/day. Data are presented as means ± SE (N = 5). ∗ P < 0.05 versus control.

The available therapy for treating primary hyperparathyroidism, a disease that so far has resisted pharmacologic intervention, has been limited to surgical removal of the affected glands. Here, we showed that parathy-

roidectomy in rats results in marked reduction of serum PTH levels and hyperphosphatemia, a well-known symptom in hypoparathyroidism [23–25]. In addition, parathyroidectomy induced the concomitant decrease of renal

van Abel et al: PTH-dependent regulation of renal TRPV5

1717

Fig. 5. Immunofluorescence localization of the Ca2+ -sensing receptor and transient receptor potential, vanilloid, member 5 (TRPV5). Double staining of a mice kidney cortex section showing staining for Ca2+ sensing receptor (A) and TRPV5 (B). The asterisks indicate the tubular segments, where Ca2+ -sensing receptor and TRPV5 colocalize. The tubules depicted by the arrowheads stained intensely for Ca2+ -sensing receptor, but were negative for TRPV5. G is glomerulus. Colocalization of Ca2+ -sensing receptor (C) and TRPV5 (D) in distal convoluted tubule (DCT) and connecting tubule (CNT) of kidney cortex section.

mRNA and protein abundance of TRPV5, calbindinD 28K , and NCX1. As demonstrated by the hypercalciuria present in TRPV5 knockout mice, the decline in renal expression of transcellular Ca2+ transport proteins in the present experiment is indicative for a decreased capacity of active Ca2+ reabsorption [26] and relates to the development of the observed hypocalcemia. Serum PTH levels tended to increase after PTH supplementation, which was apparently sufficient to restore the expression of the renal Ca2+ transport proteins and increase serum Ca2+ concentrations. Moreover, these findings imply that PTH, in concert with its well-established effect on Ca2+ resorption from bone [27], increases renal Ca2+ reabsorption by up-regulating the expression of TRPV5 and other Ca2+ transport proteins, thereby maintaining the Ca2+ balance. In agreement with this finding, PTH receptors have been detected throughout the kidney, as well as in the actively Ca2+ transporting tubules DCT and CNT [9]. Thus, PTH is capable of stimulating active Ca2+ transport in kidney through receptor-mediated actions. Calcimimetic compounds provide a novel non-surgical therapy for hyperparathyroidism by enhancing the sensitivity of the Ca2+ -sensing receptor to extracellular Ca2+ , thereby suppressing the secretion of PTH and normalizing serum Ca2+ levels [11]. Studies of inherited disorders caused by mutations in Ca2+ -sensing receptor clearly suggested that this receptor is the mechanism for Ca2+ mediated regulation of PTH secretion [28]. To determine

whether NPS R-467 could indeed effectively lower PTH levels and affect renal Ca2+ handling, mice were treated for 7 days with the calcimimetic compound NPS R-467. In agreement with previous findings, NPS R-467 successfully decreased serum PTH levels [12, 29]. In addition to the suppression of PTH, serum Ca2+ concentrations were reduced after NPS R-467 treatment, whereas serum phosphate levels were increased. Interestingly, a similar concurrent down-regulation of the expression of the renal Ca2+ transport proteins (i.e., TRPV5, calbindin-D 28K and calbindin-D 9K , NCX1, and PMCA1b) was observed in kidneys of NPS R-467–treated mice as for the parathyroidectomized rats, and indicative for a decreased capacity of active Ca2+ reabsorption. This finding is consistent with complementary findings in other studies. A shortterm use of the type II calcimimetic compound NPS R568 was previously associated with an increase in urinary Ca2+ excretion in hypercalcemic patients with primary hyperparathyroidism [30]. Recently, Fox et al [abstract; Fox J et al, J Am Soc Nephrol SA-FC179, 2003] showed that injections with NPS R-467 in rats increased the fractional excretion of Ca2+ . Taken together, our findings suggested that PTH stimulates the coordinated expression of transcellular Ca2+ transport proteins in kidney, which could contribute to increased Ca2+ reabsorption and positively affect Ca2+ balance. Active Ca2+ reabsorption is a primary target for the regulation by calciotropic hormones. We have previously

Ca2+ reabsorption, nmol.h-1 .cm-2

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Fig. 6. Effect of parathyroid hormone (PTH) and ruthenium red on Ca2+ transport and expression levels of genes encoding Ca2+ transport proteins in primary cultures of rabbit kidney connecting tubule/cortical collecting duct (CNT/CCD). (A) Transepithelial Ca2+ transport across confluent monolayers was measured in the absence or presence of PTH and ruthenium red (RR). At the end of the incubation period apical medium was collected to determine the amount of Ca2+ transported across the monolayer. Using real-time quantitative polymerase chain reaction (PCR), mRNA expression of transient receptor potential, vanilloid, member 5 (TRPV5) (B), calbindin-D 28K (C), and Na+ /Ca2+ -exchanger (NCX1) (D), of the different experimental groups were measured and presented as a ratio to hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression. (E) Western blot analysis of homogenates from rabbit CNT/CCD cultured in the absence or presence of PTH and ruthenium red, labeled with antibodies against calbindin-D 28K and Na+ -K+ -ATPase, as an internal control for equal loading. (F) Densitometric analysis of the corresponding blots. Calbindin-D 28K expression is presented as a ratio to Na+ /K+ -ATPase expression in relative percentages. Control, unstimulated cells; PTH, cells stimulated with 100 nmol/L PTH for 120 hours; cultured in the absence () or presence (
) of 10 lmol/L ruthenium red. Data are presented as means ± SE (N = 9). # P < 0.05 versus all; ∗ P < 0.05 versus control in the presence and absence of ruthenium red; ‡ P < 0.05 versus control in the absence of ruthenium red.

25

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1718 van Abel et al: PTH-dependent regulation of renal TRPV5

van Abel et al: PTH-dependent regulation of renal TRPV5

shown that the expression of TRPV5, as well as the other proteins involved in active Ca2+ transport, is positively regulated by 1,25(OH) 2 D 3 and other hormones, like estrogens [19, 31, 32]. 1,25(OH) 2 D 3 is synthesized primarily as a result of the conversion of 25-hydroxyvitamin D 3 by the renal enzyme 25-hydroxyvitamin D 3 -1a-hydroxylase (1a-OHase). PTH is the major regulator of renal 1aOHase [33]. In turn, 1,25(OH) 2 D 3 controls parathyroid gland growth and suppresses the synthesis and secretion of PTH. Moreover, 1,25(OH) 2 D 3 and its analogues are frequently used as treatment for secondary hyperparathyroidism [34, 35]. Therefore, 1,25(OH) 2 D 3 could be responsible for the observed effect on the expression of Ca2+ transport proteins. However, the decline in TRPV5, calbindin-D 28K and calbindin-D 9K and NCX1 expression after NPS R-467 treatment was not due to decreased levels of 1,25(OH) 2 D 3 . In contrast, the decrease in serum PTH levels in the parathyroidectomized rats did result in reduced 1,25(OH) 2 D 3 levels. However, PTH supplementation did not alter serum 1,25(OH) 2 D 3 levels, but was sufficient to concomitantly elevate the expression of TRPV5, calbindin-D 28K , and NCX1. This supports a regulatory mechanism primarily controlled by PTH. However, a possible mechanism by which PTH could affect expression is an increased sensitivity for 1,25(OH) 2 D 3 via elevated vitamin D receptor expression. 1,25(OH) 2 D 3 exerts its biologic actions by binding to the vitamin D receptor and the response in the target tissues to 1,25(OH) 2 D 3 is directly related to the vitamin D receptor abundance [33]. Parathyroidectomy, PTH supplementation, or NPS R-467 infusions did, however, not change vitamin D receptor mRNA levels, suggesting that the alterations in renal Ca2+ transport protein expression were not associated with changes in renal vitamin D receptor expression. Together, these findings imply that PTH stimulates renal Ca2+ reabsorption by up-regulation of the expression of TRPV5 and other Ca2+ transport proteins, independently of 1,25(OH) 2 D 3 . In addition to the presence in parathyroid glands, the Ca2+ -sensing receptor is also expressed in other cells involved in systemic Ca2+ homeostasis. Notable among these are certain epithelial cells in kidney cortex, particularly those in the cortical thick ascending limb (TAL) and DCT [36]. Therefore, the observed effect on serum Ca2+ levels after NPS R-467 infusion could also be explained by a direct action of the Ca2+ -sensing receptor on the Ca2+ transport mechanisms in kidney. Immunohistochemical analysis of kidney cortex sections confirmed the intense basolateral expression of Ca2+ -sensing receptor in TAL. Moreover, we demonstrated that the Ca2+ -sensing receptor is present in DCT and CNT, confirming previous observations [10, 36]. However, the acute hypocalcaemic response to the type II calcimimetic compound NPS R568 was abolished in (thyro) parathyroidectomized rats infused with Ca2+ or PTH, suggesting that the effects of

1719

the calcimimetic compounds are totally dependent on the parathyroid Ca2+ -sensing receptor–mediated inhibition of PTH secretion [29, 37]. In addition, pharmacologic evidence from a study using NPS R-467 in rats suggested that renal Ca2+ -sensing receptor contributes to the regulation of Ca2+ reabsorption by modulating the response to PTH rather than by affecting transport mechanisms directly [abtstract; Fox, et al, J Am Soc Nephrol SA-FC179, 2003]. Nevertheless, in the present study, the calcimimetic compound NPS R-467 could directly affect serum Ca2+ levels via inhibitory actions on the renal Ca2+ -sensing receptor. Remarkably, after NPS R-467 treatment Ca2+ -sensing receptor mRNA levels were down-regulated, which could indeed mediate the decreased expression of Ca2+ transport proteins by directly affecting expression or by modulating the effect of PTH [38, 39]. However, parathyroidectomy in rats did not change the Ca2+ -sensing receptor mRNA levels, suggesting that differences in Ca2+ -sensing receptor expression are not entirely responsible for the observed effect on renal Ca2+ transport proteins. In addition to abundance, the activity of the Ca2+ -sensing receptor could play a role in directly affecting Ca2+ transporter expression. It is unlikely that Ca2+ sensing receptors on parathyroid cells and kidney cells are exposed to different levels of NPS R-467. However, the cellular environment in which the receptor is expressed could profoundly influence its response to ligands. This has already been established for G protein–coupled receptors as well as for the different effects of calcimimetics on PTH and calcitonin secretion [37, 40]. Studies of inherited disorders caused by mutations in Ca2+ -sensing receptor clearly suggest that this receptor is the mechanism for Ca2+ -mediated regulation of both PTH secretion and Ca2+ excretion by the kidney [28]. However, no data are present about the physiologic function of the Ca2+ -sensing receptor in DCT/CNT and its possible effect on Ca2+ reabsorption. Our findings suggest that there are additional or alternative mechanisms to account for the altered renal Ca2+ handling, involving PTH-mediated regulation of the expression of renal Ca2+ transport proteins. In various studies exploring the regulatory role of 1,25(OH) 2 D 3 , estrogens, PTH, and dietary Ca2+ , we observed the concomitant up-regulation of Ca2+ transport proteins in kidney [19, 32]. Likewise, genetic ablation of TRPV5 in mouse resulted in a decreased expression of calbindin-D 28K and NCX1. As transcellular Ca2+ reabsorption in DCT/CNT was abolished in these TRPV5 knockout mice, the concomitant decrease in Ca2+ transport proteins, even in the presence of elevated 1,25(OH) 2 D 3 levels, suggested a regulatory mechanism controlled primarily by TRPV5 [26]. We, therefore, hypothesized that the Ca2+ influx through TRPV5 predominantly controls the expression of the downstream Ca2+ transport proteins. To further

1720

van Abel et al: PTH-dependent regulation of renal TRPV5

substantiate the effect of PTH on Ca2+ influx, rabbit primary cell cultures were used. These cultures retain many characteristics of the original epithelium, including a net apical-to-basolateral Ca2+ transport and hormone responsiveness [13]. In rabbit, the CNT and CCD have been implicated in active Ca2+ reabsorption and it has been shown that the transcellular Ca2+ transport proteins are all present in these cells [5, 18, 21, 41]. Using polarized monolayers of the rabbit CNT/CCD, we demonstrated that PTH stimulates transepithelial Ca2+ transport, which was accompanied by an increased expression of TRPV5, calbindin-D 28K , and NCX1. Subsequently, the entry of Ca2+ through TRPV5 was blocked with ruthenium red, which is a potent inhibitor of TRPV5 with an inhibition constant (IC 50 ) of around 110 nmol/L [14]. Blockage of TRPV5 by ruthenium red eliminated PTH-stimulated transepithelial Ca2+ transport and simultaneously decreased the expression of calbindin-D 28K and NCX1, whereas TRPV5 expression remained unaffected. Apparently, there are two mechanisms that are responsible for the effect on calbindin-D 28K and NCX1 expression in the primary CNT and CCD cell monolayers. The magnitude of the Ca2+ influx through TRPV5 predominantly controls the expression of calbindin-D 28K and NCX1. In addition, direct stimulation by PTH results in increased expression of TRPV5, calbindin-D 28K , and NCX1. Taken together, our findings imply that the magnitude of the Ca2+ influx through TRPV5 controls the expression of the Ca2+ transport proteins. In addition, these results confirm the direct effect of PTH on the expression of these renal proteins. Moreover, they explain the coordinated regulation of the renal Ca2+ transport proteins and further emphasize the gatekeeper function of TRPV5.

ACKNOWLEDGMENTS The authors thank Dr. E. Nemeth of NPS Pharmaceuticals (Salt Lake City, UT, USA) for kindly providing the calcimimetic compound NPS R-467 and the anti- Ca2+ -sensing receptor antibody. This work was supported by grants of the Dutch Organization of Scientific Research (Zon-Mw 902.18.298, Zon-Mw 016.006.001) and the Dutch Kidney Foundation (C03.6017). Reprint requests to Ren´e J.M. Bindels, 160 Cell Physiology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. E-mail: [email protected]

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